This invention relates to solid phase synthesis of chemical compounds and more particularly to a synthesis method employing support units arranged in groups of known spatial organization of the support units.
The split synthetic method (originally called portioning-mixing) introduced by Furka and his colleagues in 1988 made it possible to prepare millions of compounds in a few days. Furka, xc3x81.; Sebestyxc3xa9n, F.; Asgedom, M, Dibxc3x3, G. In Highlights of Modern Biochemistry, Proceedings of the 14th international Congress of Biochemistry, VSP. Utrecht, The Netherland; 1988, Vol. 5, p 47; Furka, xc3x81.; Sebestyen, F.; Asgedom, M.; Dibxc3x3, G. In Abstracts of 10th International Symposium of Medicinal Chemistry, Budapest, Hungary, 1983, p 288.
The method is based on the solid phase synthetic procedure introduced by Merrifield. The new method was demonstrated with the synthesis of peptide mixtures called peptide libraries. Each cycle of the solid phase synthesis was replaced by the following operations:
(i) Portioning of the solid support into equal samples;
(ii) Coupling a different amino acid to each sample;
(iii) Mixing the samples;
These operations were repeated until the desired length of the peptides was achieved. The synthesized peptides could be used either as real mixtures after cleaving them from the support, or in tethered form by removing only the protecting groups. The latter approach took advantage of the fact that in the split synthesis only a single compound forms on each bead (and not a mixture).
Lam et al. developed a method for screening the libraries in tethered form. Lam, K. S.; Salmon, S. F.; Hersh, F. M.; Hruby, V. J.; Kazmierski, W. M.; Knapp, R. J. Nature 1991, 354, 82. The bead carrying the bioactive peptide could easily be identified by its color and, after manually picking it out, the sequence could be determined by submitting the bead to sequencing. Later the split method was applied for preparing libraries of non-peptidic small organic molecules. Since, the identification of the structure of the majority of organic compounds is not as easy as determining the sequence of peptides, different encoding methods were developed. The code attached to each bead in the course of synthesis in parallel with the product forming reactions, preserved the synthetic history of each bead. Based on the synthetic history, the identity of the compound carried by the bead could easily be deduced. Sequences of peptides (Nikolaiev et al. Pept Res. 1993, 6, 161 (1993)) and those of oligonucleotides (Brenner et al. Proc. Nati. Acad. Sd. USA , 89, 5381 (1992)) were suggested for coding. A non-sequential encoding procedure was introduced 1993 by Ohhmeyer et al. This xe2x80x9cbinaryxe2x80x9d coding method is successfully used even nowadays.
The high-throughput screening methods applied in the majority of pharmaceutical companies are based on testing individual compounds. Although the split method does produce individual compounds, the quantity present on a single bead is too low and not enough compound is produced to make multiple tests and repetitions. In addition, the identity of the compound belonging to any bead has to be identified by experimentally determining the code. Despite the high synthetic efficiency of the split method, most of the pharmaceutical companies prefer to use automated parallel synthesis methods for preparation of their combinatorial libraries. By these methods each compound is produced in several milligram quantities. The parallel synthesis, however, is slow and expensive. For this reason efforts have been made to combine the high efficiency of the split synthesis with the advantage of the parallel method in producing the new compounds in higher quantities.
The high efficiency of the split synthesis is a result of the fact that in a reaction step carried out with a single reagent, thousands or even millions of new compounds may form in microscopic beads of the solid support. In order to be able to produce the new compounds in milligram quantities, the microscopic individual beads have to be replaced by their macroscopic assemblies enclosed in permeable bags or capsules. These macroscopic synthetic units have to be redistributed among the reaction vessels before each synthetis step according to the rules of the split synthesis. In order to be able to do this, and to make possible the identification of the formed compound, the bags or capsules have to be labeled. Even in this case redistribution of thousands of bags or capsules takes too long time. In one of the methods developed to solve the problem, a microchip is enclosed into each capsule (Nicolaou et al. Angew. Chem. Int. Ed. EngI, 36, 2289. (1995)) which stores an electronic code. The capsules are pooled after each synthetic step and fed into an automatic machine constructed for sorting the capsules. In the sorting process first the electronic codes are determined, then the capsules, one at a time, are automatically delivered into their destination reaction vessels. Sorting of 10,000 capsules takes about 10 hours.
In another solution, small ceramic plates are applied, which are grafted on their surface with derivitized polystyrene. The plates are labeled by a two dimensional visual code etched by laser onto the ceramic support (Xiao et al. Angew. Chem. Int. Ed. EngI., 36,780. (1997)). Again the code is readable by the sorting machine.
The present invention is designed to preserve those operations of the split synthesis technique which are important for the high efficiency, i.e. splitting before coupling and coupling the reagent to many properly grouped macroscopic units. Mixing or pooling is omitted, since this operation is required only when working with small independent beads as solid support, to make a near homogenous mixture before the split operation. In the present invention a radically new method is introduced to put the above principles into practice.
In accordance with the method of the invention and within the framework of the split synthesis technique, compounds are synthesized on macroscopic solid support units which are spatially organized in groups, referred to herein as strings, so that the location of each unit in a string is known and recorded. Each string is placed in a reaction vessel for contact with a suitable reagent or reagents to couple a first component on the support units. Each string is submitted to a different reaction and the sequential arrangement of the support units is maintained throughout the whole reaction step and any subsequent washing steps. At the completion of a reaction step the support units of the strings (referred to as source strings) are reorganized into a different sequential arrangement to form new strings (referred to as destination strings) for the next reaction step. The reorganization of the support units into new destination strings is recorded for each reaction step in the synthesis. The destination strings may then be converted to source strings by subjecting each string to different second reaction steps to couple another component onto the solid support units the reaction and sorting steps are repeated until the desired products are formed. The sequence history is reviewed and the composition of each product is predicted without the need for tags or labels. The reorganization step, which is referred to herein as sorting, can be carried out manually or preferably by automated means.
The support units are formed of suitable materials which carry functional groups for the attachment of the first building block of the compound being formed. Methods for functionalizing materials for solid support reactions are well understood in the art. The support units may be formed into various shapes to ensure maximum contact with the reagents.
In one embodiment of the invention the support units are disk shaped with a center opening. In this embodiment the support units are retained in their position in the string by arrangement on an elongated flexible retainer, such as a string or cord, which extends through the center openings of the support units. The support units are reorganized after reaction by moving the support units in a predetermined pattern, referred to as pattern sorting, from their source strings onto new destination strings or cords. In a modification of this embodiment the support units are formed on Chiron crowns that comprise a stem and a depending crown portion. The crown portion is derivitized for coupling the first building block of the compounds being synthesized. Chiron crowns are commercially available and do not per se form a part of this invention.
In another embodiment the support units comprise conventional resin beads confined within a capsule or porous bag. The capsules and porous bags are readily adapted for easy sorting during the reorganization step.